Analytical and Bioanalytical Chemistry

, Volume 411, Issue 2, pp 439–448 | Cite as

Epitope and affinity determination of recombinant Mycobacterium tuberculosis Ag85B antigen towards anti-Ag85 antibodies using proteolytic affinity-mass spectrometry and biosensor analysis

  • Francesca Rinaldi
  • Loredana Lupu
  • Hendrik Rusche
  • Zdeněk Kukačka
  • Sara Tengattini
  • Roberta Bernardini
  • Luciano Piubelli
  • Teodora Bavaro
  • Stefan Maeser
  • Loredano Pollegioni
  • Enrica Calleri
  • Michael PrzybylskiEmail author
  • Caterina TemporiniEmail author
Research Paper


Tuberculosis (TB) is the first cause of death from infectious diseases worldwide. Only a single anti-TB vaccine is currently available for clinical use, but its efficacy is not achieved with certainty. The aim of this work is to provide a basis for the rational design of a neo-glycoconjugate vaccine against TB. Structural characterization of recombinant antigenic proteins from Mycobacterium tuberculosis (MTB) Ag85B (rAg85B, variants, and semi-synthetic glycoconjugates) was initially carried out. Identification of antibody epitope analyses by proteolytic affinity-mass spectrometry and surface plasmon resonance (SPR) biosensor analyses were performed in order to qualitatively identify and quantitatively characterize interaction structures of the antigens with antibodies from different sources. A commercial monoclonal antibody and polyclonal antibodies from different sources (patients with active TB, vaccinated individuals, and a healthy control) were employed to analyze antigen-antibody interactions. These combined approaches provided the identification of different assembled epitope regions on the recombinant MTB antigens, their affinity binding constants in the interactions with specific antibodies, and revealed the importance of protection from excessive glycosylation. The identified epitope peptides should constitute a suitable basis for the design of new specific target vaccines.

Graphical abstract


Antibody epitope identification Affinity mass spectrometry Surface plasmon resonance Ag85B Tuberculosis Glycoconjugate vaccine 



The research visiting stay of Francesca Rinaldi at the Steinbeis Centre for Biopolymer Analysis and Biomedical Mass Spectrometry, Rüsselsheim am Main, was supported by an increase of the PhD scholarship and a grant for international mobility from the University of Pavia.

We dedicate this paper to the memory of Prof. Massimo Amicosante, who made a substantial contribution sharing his knowledge and providing valuable suggestions with great humanity and professionalism.

Compliance with ethical standards

Antibody samples were blind analyzed and derived from anonymous patients belonging to a no profit Biobank located at University of Rome “Tor Vergata”.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

216_2018_1466_MOESM1_ESM.pdf (1.2 mb)
ESM 1 (PDF 1196 kb)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Francesca Rinaldi
    • 1
    • 2
  • Loredana Lupu
    • 2
  • Hendrik Rusche
    • 2
  • Zdeněk Kukačka
    • 2
  • Sara Tengattini
    • 1
  • Roberta Bernardini
    • 3
  • Luciano Piubelli
    • 4
    • 5
  • Teodora Bavaro
    • 1
  • Stefan Maeser
    • 2
  • Loredano Pollegioni
    • 4
    • 5
  • Enrica Calleri
    • 1
  • Michael Przybylski
    • 2
    Email author
  • Caterina Temporini
    • 1
    Email author
  1. 1.Department of Drug SciencesUniversity of PaviaPaviaItaly
  2. 2.Steinbeis Centre for Biopolymer Analysis and Biomedical Mass SpectrometryRüsselsheim am MainGermany
  3. 3.Department of Biology and Animal Technology StationUniversity of Rome “Tor Vergata”RomeItaly
  4. 4.Department of Biotechnology and Life SciencesUniversity of InsubriaVareseItaly
  5. 5.The Protein Factory Research CentrePolitecnico of Milan and University of InsubriaMilanItaly

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